How do you know if your antibody is working properly in immunohistochemistry (IHC)? In this Tech Tip, Abbey discusses the use of positive and negative tissue controls, isotype controls, and controls for phospho-specific and modification-specific antibodies.
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What can the right controls tell me about the performance of my antibody in immunohistochemistry, and what kind of IHC controls should I consider using? I’m Abbey, research associate in the IHC group at Cell Signaling Technology, and this is CST Tech Tips.
When you observe staining in tissues after applying an antibody in an IHC assay, that’s a good start - but you also need to know whether the staining is specific for the intended target. No single assay is sufficient to demonstrate antibody specificity and sensitivity, but performing multiple experiments including different controls can give you more confidence in your IHC results.
We’ve already covered one type of IHC controls in our Tech Tips video on Cell Pellet Controls. Click the link in the video description ( • How to incorporate cel... ) to check that out.
In this video, I’ll go over positive and negative tissue controls, isotype controls, and controls for phospho-specific and modification-specific antibodies.
If the protein of interest is known to be expressed abundantly in one tissue type, and not expressed in another, you can compare staining in these positive and negative tissues to your experimental sample. You should use the same protocol and reagents, such as antigen retrieval buffers and antibody diluent, in both the control and experimental samples.
If either the positive or negative control doesn’t give the expected result, you should troubleshoot your protocol and antibody working concentration before proceeding with your experimental samples. It’s important to check you aren’t applying your antibody at too high of a concentration, since this can lead to off-target binding.
In some cases, you may be able to obtain tissues with both positive and negative cell types present.
For example, in the heart, smooth muscle actin is present in the vascular smooth muscle cells that make up blood vessel walls, but it is not present in the surrounding cardiac muscle. In this tissue there is both a positive and negative internal control.
If absolute positive and negative tissues aren’t available, you may be able to use differential protein expression, meaning tissues with relatively higher or lower levels of protein, to gauge specificity of antibody staining.
To identify these tissues, you can consult mRNA expression profiles of tissues available from various databases, or in-situ hybridization data from the literature.
One caveat to note is that the correlation between protein and RNA expression varies by target.
An isotype control is a type of negative control. You should choose the same isotype and apply at the same concentration as your experimental antibody.
If you do observe staining with an isotype control, this indicates at least some of the signal from the primary antibody is not specific.
This could be caused by Fc receptor mediated interactions, sometimes called backwards binding, or general stickiness of your incubation conditions. If you’re using biotin-based detection, endogenous biotin in the tissue can also contribute.
Phospho-specific antibodies detect proteins only when they are phosphorylated. In addition to the controls I mentioned earlier, you can treat your tissue sample with phosphatase to remove phosphate groups and test the phospho-specificity of your antibody. You should expect to see complete abolishment of staining in phosphatase treated tissues.
To assess modification specificity when using methyl, acetyl, non-phospho, and cleavage specific antibodies, you can design IHC control experiments using blocking peptides. Note that blocking with a single peptide alone doesn’t confirm specificity. For a true test of modification specificity, compare the staining in the presence of the non-modified peptide versus peptide with the modification.
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